- Biofuel Research Journal

Biofuel Research Journal 9 (2016) 372-378
Journal homepage: www.biofueljournal.com
Original Research Paper
Fungal biomass and ethanol from lignocelluloses using Rhizopus pellets under simultaneous
saccharification, filtration, and fermentation (SSFF)
Somayeh FazeliNejad, Jorge A. Ferreira*, Tomas Brandberg, Patrik R. Lennartsson, Mohammad J. Taherzadeh
Swedish Centre for Resource Recovery, University of Borås, SE 501 90, Borås, Sweden.
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GRAPHICAL
ABSTRACT
HIGHLIGHTS
nd
 Economically viable production of 2 generation
bioethanol cannot rely on a single product.
SSFF can be used for production of ethanol and
biomass from wheat straw.
Glucose present in the feed controlled the
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assimilation of xylose and acetic acid.
The fungal growth rate was found not to be
influenced by the feed composition.
Rhizopus biomass yields of up 0.34 g/g and ethanol
yields of 0.40 g/g were obtained.
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ARTICLE INFO
ABSTRACT
Article history:
Received 23 October 2015
Received in revised form 5 January 2016
Accepted 6 January 2016
Available online 1 March 2016
The economic viability of the 2nd generation bioethanol production process cannot rely on a single product but on a biorefinery
built around it. In this work, ethanol and fungal biomass (animal feed) were produced from acid-pretreated wheat straw slurry
under an innovative simultaneous saccharification, fermentation, and filtration (SSFF) strategy. A membrane unit separated
the solids from the liquid and the latter was converted to biomass or to both biomass and ethanol in the fermentation reactor
containing Rhizopus sp. pellets. Biomass yields of up to 0.34 g/g based on the consumed monomeric sugars and acetic acid
were achieved. A surplus of glucose in the feed resulted in ethanol production and reduced the biomass yield, whereas limiting
glucose concentrations resulted in higher consumption of xylose and acetic acid. The specific growth rate, in the range of
0.013-0.015/h, did not appear to be influenced by the composition of the carbon source. Under anaerobic conditions, an
ethanol yield of 0.40 g/g was obtained. The present strategy benefits from the easier separation of the biomass from the
medium and the fungus ability to assimilate carbon residuals in comparison with when yeast is used. More specifically, it
allows in-situ separation of insoluble solids leading to the production of pure fungal biomass as a value-added product.
Keywords:
Cellulosic ethanol
Animal feed
Rhizopus sp.
SSFF
Wheat straw
© 2016 BRTeam. All rights reserved.
* Corresponding author at: Tel.: +46-33-4354638
E-mail address: [email protected]
Please cite this article as: FazeliNejad S., Ferreira J.A., Brandberg T., Lennartsson P.R., Taherzadeh M.J. Fungal protein and ethanol from lignocelluloses
using Rhizopus pellets under simultaneous saccharification, filtration and fermentation (SSFF). Biofuel Research Journal 9 (2016) 372-378.
DOI: 10.18331/BRJ2016.3.1.7
373
FazeliNejad et al. / Biofuel Research Journal 9 (2016) 372-378
1. Introduction
Nowadays, the production of 1st generation bioethanol from agricultural
sugar- or starch-rich crops, as a replacement to gasoline, is well established at
commercial scale. The leading ethanol-producing countries, USA and Brazil,
use corn and sugarcane as main feedstocks, respectively (RFA, 2014).
However, ethical issues related to the use of sugar- or starch-rich feedstocks
for fuel production instead of being directed to human consumption have put
pressure on finding alternative feedstocks (Cherubini, 2010).
The production of ethanol from lignocellulosic materials has been
considered for several decades (Leonard and Hajny, 1945). Nevertheless, due
to their recalcitrant structure, a feasible commercial facility producing the socalled 2nd generation bioethanol is presently inexistent and is only limited to
some pilot plants (Pandey et al., 2015). Constraints include the cost-intensive
pretreatment needed to open up the lignocellulosic structure, the cost of
enzymes needed in the post-pretreatment stage, the lack of robust
microorganisms that can cope with inhibitors, and robust cultivation strategies
that can meet all requirements for feasible 2nd generation bioethanol prodution
(Ishola, 2014). Another conclusion drawn by the intensive studies conducted
over years is that a facility using lignocelluloses as feedstock cannot rely on a
single product (i.e., ethanol) for achieving an economically-viable operation
(Pandey et al., 2015).
The most commonly used strategies for production of ethanol include
simultaneous saccharification and fermentation (SSF) and separate hydrolysis
and fermentation (SHF). Running a SSF instead of a SHF circumvents the
product inhibition of cellulase enzymes due to glucose accumulation
(Olofsson et al., 2008). However, SSF disadvantageously requires the use of
new microorganisms at each batch since it is difficult to separate them from
the medium (Wingren et al., 2003). Therefore, a new cultivation strategy, i.e.,
simultaneous saccharification, filtration, and fermentation (SSFF) was
developed by Ishola et al. (2013). This new concept consists of a membrane
unit (cross-flow membrane) connecting a hydrolysis reactor to a fermentation
reactor. The enzyme-slurry mixture from the hydrolysis reactor is filtered and
the sugar-rich permeate is continuously supplied to the fermentation reactor
while the residues are returned to the hydrolysis reactor. The fermented
medium in the fermentation reactor is also pumped back to the hydrolysis
reactor for volume balance (Fig. 1).
and there is also the possibility to reuse the fermenting cells. The fact that
the fermenting cells is free from solid substrates (i.e., lignocellulosic
materials) in the fermentation reactor under the SSFF, opens up the
opportunity for the production of a second value-added product (i.e.,
biomass as animal feed) from the residual glucose, pentose sugars (such
as xylose), and other components (such as acetic acid) contained in the
pretreated wheat straw slurry. This would be a similar situation as exists
for the 1st generation bioethanol plants from starch grains where both
ethanol and animal feed products are generated (Kim et al., 2008).
Edible filamentous fungi have been previously used for production of
protein-rich biomass (animal feed) from various types of substrates
(Ferreira et al., 2013). For instance, tempe-isolated zygomycete Rhizopus
sp. has been found to cope with inhibitors contained in lignocellulosic
hydrolysates (FazeliNejad et al., 2013). Moreover, these microorganisms
form pellets, and therefore, could be easily separated from the medium
(Nyman et al., 2013). They also consume pentose sugars contrary to
baker’s yeast Saccharomyces cerevisiae (Wikandari et al., 2012). These
attributes mark these fungi as appropriate candidates for application in the
SSFF. Among the different lignocellulosic materials, wheat straw is an
economic agricultural by-product available at huge amounts with
potentials for production of ethanol in view of its cellulose (33%) and
hemicellulose (33%) dry weight contents (Canilha et al., 2006). It is worth
quoting that various studies have been carried out on the pretreatment and
hydrolysis of wheat straw (Talebnia et al., 2010; Baboukani et al., 2012;
Peng et al., 2012).
In the present work, SSFF was used for ethanol production from
glucose using S. cerevisiae as well as biomass (animal feed) production
from residual carbon sources using Rhizopus sp. in pellet form. The
effects of the enzyme addition to the hydrolysis reactor, temperature in the
hydrolysis and fermentation reactors, and aeration for assimilation of
carbon sources in the filtered permeate for production of biomass (animal
feed) were investigated. This is the first work reporting the use of SSFF
with filamentous fungi Rhizopus sp. for the production of two value-added
products, i.e., bioethanol and animal feed from lignocellulosic materials.
2. Materials and methods
2.1. Microorganisms
Rhizopus sp. CCUG 61147 (Culture Collection University of
Gothenburg, Sweden) isolated from Indonesian leaves traditionally used
for preparation of tempe, was used in this work. The strain was identified
as RM4 in a previous publication (Wikandari et al., 2012). The fungus
was kept in Potato Dextrose Agar (PDA) plates and its incubation and
preparation for inoculation were carried out according to FazeliNejad et
al. (2013). A strain of S. cerevisiae, Ethanol Red, kindly provided by
Fermentis (France) in dry form was also used.
2.2. Pretreated wheat straw slurry
Slurry of wheat straw delivered by SEKAB E-Technology
(Örnsköldsvik, Sweden) was produced by continuous treatment of wheat
straw at 22 bar for 5-7 min. The resulting slurry, a liquid fraction with fine
particles, had 14.6% suspended solids (SS) and 23.8% total solids (TS).
The liquid had a pH value of 2.0 and contained (in g/L): glucose, 7.2;
xylose, 22.1; galactose, 2.3; arabinose, 4.6; acetic acid, 5.9; 5-hydroxy
methyl-furfural (HMF), 2.1; and furfural, 4.2. The solid fraction contained
34.7% (w/w) glucan and 4.6% (w/w) xylan.
2.3. Enzyme cocktail
Cellic® CTec2, kindly provided by Novozymes (Denmark), was used
in the experiments for enzymatic treatment. The product had an activity of
168 filter paper units (FPU)/mL.
Fig.1. Schematic representation of SSFF for aerobic biomass (Rhizopus sp.) production.
2.4. Inoculant preparation and cultivations in shake-flasks
Therefore, SSFF combines the advantages of both SSF and SHF by
simultaneously solving their limitations, i.e., both hydrolysis and fermentation
can be carried out at optimal conditions, the end-product inhibition is avoided,
A complex medium containing (in g/L): xylose, 20; potato extract, 4;
soybean peptone, 6; and CaCO3, 6 was used for preparation of Rhizopus
sp. pellets as inoculant. The medium (50 mL) was transferred to 250 mL
Please cite this article as: FazeliNejad S., Ferreira J.A., Brandberg T., Lennartsson P.R., Taherzadeh M.J. Fungal protein and ethanol from lignocelluloses
using Rhizopus pellets under simultaneous saccharification, filtration and fermentation (SSFF). Biofuel Research Journal 9 (2016) 372-378.
DOI: 10.18331/BRJ2016.3.1.7
.
374
FazeliNejad et al. / Biofuel Research Journal 9 (2016) 372-378
cotton-plugged Erlenmeyer flasks followed by sterilization in an autoclave at
121 °C for 20 min. It should be noted that xylose was autoclaved separately.
After mixing and inoculation with 1.0 × 10 5 spores/mL of Rhizopus sp., the
flasks were kept in a water-bath at 30 °C and 150 rpm for 72 h. The produced
Rhizopus sp. pellets were transferred to new cultivations to a cell
concentration of 1.65 ± 0.10 g/L (dry weight ± 1 SD). The new cultivations
were carried out in 250-mL Erlenmeyer flasks containing 100 mL of the same
medium which also consisted of (in g/L): (NH4)2SO4, 7.5; KH2PO4, 3.5;
CaCl2.2H2O, 1; MgSO4.7H2O, 0.75; and one of the following carbon sources
namely acetic acid, 5.0; ethanol, 10; glucose, 10; lactic acid, 10; and xylose,
10. The cultivations were kept in a water-bath at 30 °C while being shaken at
150 rpm.
With similar preparation of the initial inoculant, 2.20 ± 0.12 g/L pellets
(dry weight, ± 1 SD) were transferred to a new medium and cultivated as
described above but a combination of the carbon sources at 3.5 g/L was used.
This experiment was carried out in duplicate.
Liquid samples were withdrawn and stored at -20 °C for subsequent
analysis. At the end of the cultivation, the pellets were harvested using a
sieve, washed with distilled water, and dried in an oven at 70 °C to constant
weight for 24 h. The cultivations using single-carbon sources or their
combination were performed in quadruplicate and duplicate, respectively.
2.5. Cultivations under SSFF
The SSFF as previously described by Ishola et al. (2013) was employed.
The lignocellulosic feedstock was hydrolysed enzymatically in a separate
vessel (hydrolysis reactor) and the resulting sugar-rich liquid was circulated
through the fermentation reactor, where the fungal biomass production took
place. The solid fraction was separated from the sugar-rich stream by a crossflow membrane. However, a cell retention system was not needed in this
work since 5 mm spherical pellets of Rhizopus sp. were used and they
maintained this morphology throughout the cultivations.
For the SSFF trials, pellets were prepared as described above and
transferred to a 750 mL fermentor (Ant, Belach Bioteknik AB, Sweden)
containing sterilized salt solution as described above and 0.1 g/L antifoam.
The transferred Rhizopus sp. pellets had an initial dry weight within the range
1.5-2.1 g and the volume was adjusted to a total volume of 500 mL. Wheat
straw slurry was transferred to a parallel hydrolysis reactor (Memma, Belach
Bioteknik AB, Sweden) and diluted with deionized water to 5.0% SS to a
total volume of 3.5 L. The salt and antifoam content was the same as in the
fermentation reactor. The cross-flow membrane unit was set up according to
Ishola et al. (2013). After integration (Fig. 1), the flow of the filtrate through
the fermentation bioreactor was 40 mL/h.
In a first experiment, the integration of the SSFF system was preceded by
24 h enzymatic decomposition by addition of Cellic ®CTec2, corresponding to
10 FPU/g SS. The pH was initially adjusted to 5.5 in both reactors and
regulated to 5.5 in the fermentation reactor by on-line addition of 2.0 M
NaOH. The temperature was kept at 50 °C in the hydrolysis reactor and 35 °C
in the fermentation reactor. The stirring was 350 rpm in the hydrolysis vessel
and 100 rpm in the fermentation bioreactor, which was aerated at 1 vvm
(volume of air per volume of liquid per minute). The experiment was carried
out in duplicate where the integration phase lasted for 140 h and 168 h.
Samples were withdrawn directly from the tubes channeling medium in and
out of the fermentation vessel. The final cell (biomass) content was analysed
by weighing it after drying at 70 °C for 24 h. The experiment was then
repeated with the same parameters but with the following differences; no
enzyme was added and the integration phase lasted for 72 h. This was
intended to investigate the impact of enzyme addition into the hydrolysis
reactor.
In a similarly initiated experiment, the effect of aeration in the
fermentation reactor was investigated by switching off the air supply after 72
h. Moreover, 10 FPU/g SS enzyme was simultaneously added into the
hydrolysis reactor. This anaerobic fermentation phase lasted for 94 h.
Another SSFF trial was also carried out where the temperature was
adjusted to 35 °C in both reactors. Enzyme (10 FPU/g SS) and 15 g of dry
baker’s yeast were added to the hydrolysis reactor. The experiment was
carried out in duplicate where the integration phase lasted for 96 and 120 h.
In a different set-up, the similar cultivation as of above (i.e., 35 °C, 10
FPU/g SS, and 15 g dry yeast) was initially performed without any integration
for 54 h. The resulting fermented slurry was then distilled using a rotary
evaporator (Labinett, Sweden) at 140 °C (oil bath), and 30 rpm rotation
speed at atmospheric pressure. The water content lost during distillation
was re-adjusted by the addition of sterile ultrapure water. The resulting
slurry, now without ethanol, was used for integration with the SSFF and
aerobic production of Rhizopus sp. biomass as described above during 96
h.
2.6. Analytical methods
The measurements of glucose, metabolites, and inhibitors
concentrations as well as spore counting were performed according to
FazeliNejad et al. (2013). The SS was determined by filtration with
Munktell filters, Grade 3 (5-8 µm) while the TS were determined by
drying the samples to a constant weight at 105 °C overnight. The solid
fraction of the wheat straw slurry and the enzyme activity were analyzed
according to the NREL protocols (Adney and Baker, 2008; Sluiter et al.,
2011).
3. Results and discussion
3.1. SSFF of wheat straw slurry with Rhizopus sp. pellets
Production of additional products in a biorefinery concept has been
proposed to improve the process economy of ethanol production from
cellulosic raw materials (Wheals et al., 1999; Gnansounou and Dauriat,
2010). Animal feed in the form of Rhizopus sp. biomass has been
suggested as a valuable co-product for ligno-ethanol in the present study.
Implementing SSFF for aerobic production of Rhizopus sp. biomass
entails the application of a cross-flow membrane to separate available
sugars and other organic compounds from a pretreated lignocellulosic
slurry (Fig. 1). The filtrate is supplied to an aerated fermentor, where
carbon sources are consumed by Rhizopus sp. pellets in order to produce
biomass (animal feed).
The pellet morphology is useful in order to prevent leakage of biomass
when liquid is pumped back to the hydrolysis reactor. This reflux is
necessary in order to maintain the liquid balance between the vessels and
to prevent increasing the dry matter content of the slurry. Besides, the
glucose concentration, which would increase as a result of enzymatic
decomposition of cellulose and could inhibit the enzymes, can be
controlled in this way.
On the other hand, the filtration of the slurry is in itself a very
important operation since the solid fraction must not be mixed with the
biomass, which would result in a downstream separation problem. In
addition to biomass, the Rhizopus sp. used in this work is also a potential
producer of ethanol (Wikandari et al., 2012).
The implementation of SSFF for production of ethanol and biomass
includes the use of continuous cross-flow membrane as described earlier.
The results obtained revealed that the filtration unit was used for up to 168
h without regeneration of the membrane and without any fouling. In a
similar experiment, involving the slurry of pretreated spruce, the same
operation was performed during 28 d without interruption, regeneration,
or fouling (Ishola et al., 2013).
3.2. Specific growth rate and biomass yield
Various SSFF experiments with Rhizopus sp. production from wheat
straw slurry were carried out in order to validate this concept. The main
difference between the different trials was the composition of the
substrate, notably its glucose content. Enzymatic decomposition of
cellulose in the solid fraction prior to integration with SSFF (Fig. 2)
produced a relatively high initial glucose concentration in contrast to a
similar experiment without enzyme addition (Table 1).
The addition of baker’s yeast to the hydrolysis vessel in a different
experiment nearly eliminated the glucose in the inflow to the fermentation
reactor. Furthermore, an experiment was carried out where the amount of
glucose was reduced by the addition of baker’s yeast and the produced
ethanol was also removed by distillation. The resulting mix was used for
Rhizopus sp. production by SSFF (Table 1).
Please cite this article as: FazeliNejad S., Ferreira J.A., Brandberg T., Lennartsson P.R., Taherzadeh M.J. Fungal protein and ethanol from lignocelluloses
using Rhizopus pellets under simultaneous saccharification, filtration and fermentation (SSFF). Biofuel Research Journal 9 (2016) 372-378.
DOI: 10.18331/BRJ2016.3.1.7
.
375
FazeliNejad et al. / Biofuel Research Journal 9 (2016) 372-378
Fig.2. Concentrations of ethanol, acetic acid, glucose, and xylose in SSFF for aerobic production of Rhizopus sp. biomass and ethanol. The integration (connection between the hydrolysis reactor and
bioreactor by a filtration unit) was preceded by 24 h of enzymatic hydrolysis. Closed symbols denote concentrations in the ingoing feed to the bioreactor (number 4 in Fig. 1), open symbols denote
concentrations in the recirculation feed from the bioreactor to the hydrolysis reactor. The expressions “in” and “out” represent the medium going from the filtration unit into the bioreactor and the
medium that leaves the bioreactor to the hydrolysis reactor, respectively, whereas “2” stands for the replicate 2 of the experiment.
The specific growth rate, µ (/h), was calculated according to the following
equation (Eq. 1):
Eq. 1
Where x0 denotes the initial biomass concentration and x is the biomass
concentration after the elapsed time t. Assuming a constant µ is debatable
because of the dynamic conditions such as substrate concentrations and the
fact that the results are sensitive to the accuracy of wet weight measurements
of the initial biomass. Growth in the form of pellets is also known to be
different from that of free cells (Metz and Kossen, 1977), but considering the
small size of the used pellets, this effect can be assumed to be relatively low.
However, the results tabulated in Table 1 show that µ as measured, hardly
changed corresponding to the substrate composition, i.e., 0.013/h < µ <
0.015/h under aerobic conditions. It could be concluded that the growth rate
showed no tendency to be affected by glucose concentrations as long as
alternative carbon sources such as acetic acid, ethanol, and xylose were
present. However, more efficient aeration could have resulted in a higher
growth rate.
The biomass yields reported in Table 1 ranged between 0.24 and 0.34
g/g consumed monomeric sugars (arabinose, galactose, glucose, and
xylose), acetic acid, and ethanol, except for the case with enzymatic
hydrolysis (i.e., high initial glucose concentration, Fig. 2) where the
biomass yield dropped due to ethanol production. These biomass yields
were in harmony with the yield obtained in separate batch experiments
with synthetic medium containing individual carbon sources (10 g/L of
each compound except for acetic acid; 5 g/L). The measured yields of
biomass for acetic acid, ethanol, and xylose in these trials were 0.30 g/g,
0.30 g/g, and 0.29 g/g, respectively, after 140 h batch cultivation (96 h for
acetic acid, data not shown). The corresponding consumption of glucose
was faster (less than 42 h), but the resulting biomass yield was as low as
Please cite this article as: FazeliNejad S., Ferreira J.A., Brandberg T., Lennartsson P.R., Taherzadeh M.J. Fungal protein and ethanol from lignocelluloses
using Rhizopus pellets under simultaneous saccharification, filtration and fermentation (SSFF). Biofuel Research Journal 9 (2016) 372-378.
DOI: 10.18331/BRJ2016.3.1.7
.
376
FazeliNejad et al. / Biofuel Research Journal 9 (2016) 372-378
Table 1.
Overview of SSFF trials including: (1) integration preceded by 24 h of enzymatic hydrolysis; (2) no addition of enzyme to the hydrolysis reactor; (3) integration preceded by 54 h of enzymatic
hydrolysis and ethanol production with yeast in the hydrolysis reactor followed by evaporation of the ethanol; (4) Enzyme and yeast were added to the hydrolysis reactor with no ethanol evaporation
before integration; (5) The air supply to the bioreactor was switched off at t = 72 h with concomitant addition of enzymes to the hydrolysis reactor. The glucose column refers to the concentration of
glucose in the bioreactor and how it developed (the uptake of arabinose and galactose are not reported).
SSFF trial
(1) Enzyme addition
Glucose
(g/L)
YE/S
(g/g)
Cultivation time
(h)
YX/S
(g/g)
140
0.11
a
168
0.14
a
~ 27 decreases to ~ 6
(2) No enzyme addition
2.2 decreases to 0.2
72
0.32
a
(3) SSF & evaporation b
2.2 to ND
96
0.34
a
96
0.24
a
(4) Yeast in hydrolysis c
<0.3
120
0.30
a
94
0.034
(5) Anaerobic with
enzyme addition
peaks at 17.5
d
µ
(/h)
Distribution of uptake (%)
Glucose
Xylose
Ethanol
Acetic acid
0.21
a
0.013
86
9
-
6
0.14
a
0.014
80
12
-
7
0.015
46
43
-
12
0.015
28
39
-
25
0.015
<1
53
22
25
0.013
1
37
28
34
0.002
95
5
-
<1
-
0.10
a
Cons.
0.40
d
YX/S = yield (g of fungal biomass/g of consumed carbon source)
YE/S = yield (g of ethanol/g of consumed carbon source)
Biomass and ethanol yields related to consumed amounts of acetic acid, arabinose, ethanol, glucose, galactose, and xylose.
This treatment resulted in reduced amounts of glucose and ethanol prior to SSFF integration.
c
This method sharply reduced the glucose content in the flux into the bioreactor.
d
Biomass and ethanol yields related to consumed amounts of glucose and xylose.
“Cons.” = consumed
“ND” = not detected.
a
b
0.11 g/g due to formation of ethanol and glycerol (data not shown),
confirming overflow metabolism (Crabtree effect) for Rhizopus sp. (Millati et
al., 2005; Lennartsson et al., 2009). The pooled standard deviation for the
biomass yields was 0.042 (± 1 SD).
3.3. Steering the uptake of carbon sources
In a separate experiment with synthetic medium, the uptake pattern was
studied in a cultivation, where acetic acid, ethanol, glucose, lactic acid, and
xylose were added to the same cultivation of Rhizopus sp. in aerobic shakeflasks. The results showed a relatively rapid consumption of glucose,
followed by acetic acid, whereas xylose and ethanol with similar consumption
trends were not totally consumed after 72 h of cultivation (Fig. 3). Lactic acid
frequently occurs as an undesired metabolite produced by contaminants
(Skinner and Leathers, 2004) and its uptake by other zygomycetes is
documented (Ferreira et al., 2013). However, no measurable consumption
of lactic acid by the Rhizopus sp. strain was confirmed in this experiment.
It is observed that the preference of carbon source, among those
examined, under the examined conditions can be ranked as follows:
glucose > acetic acid > xylose & ethanol (Fig. 3). The measured specific
growth rate, µ, was 0.013/h, i.e., similar to the level in the SSFF
experiments with wheat straw hydrolysate (Table 1), but it is difficult to
differentiate the effects of inhibitors and different aeration rates.
In the SSFF experiment with cellulase addition, it is clearly visible that
the glucose uptake was relatively efficient but had no visible positive
effects on the specific growth rate. Instead, ethanol was produced in a
respire-fermentative pattern (Fig. 2 and Table 1). In a biorefinery
context, it is probable that glucose, if available, would be used for other
Fig.3. Concentration profiles of glucose, acetic acid, xylose, ethanol, lactic acid, and glycerol in an aerated shake-flask experiment inoculated with Rhizopus sp. pellets. At the beginning of the
cultivation, the concentration of all mixed components except glycerol, which was produced during cultivation, was 3.5 g/L. Error bars denote ±1 SD.
Please cite this article as: FazeliNejad S., Ferreira J.A., Brandberg T., Lennartsson P.R., Taherzadeh M.J. Fungal protein and ethanol from lignocelluloses
using Rhizopus pellets under simultaneous saccharification, filtration and fermentation (SSFF). Biofuel Research Journal 9 (2016) 372-378.
DOI: 10.18331/BRJ2016.3.1.7
.
377
FazeliNejad et al. / Biofuel Research Journal 9 (2016) 372-378
Fig.4. Concentrations of ethanol, acetic acid, glucose, and xylose in the SSFF for aerobic production of biomass followed by anaerobic fermentation by Rhizopus sp. The air supply to the bioreactor
was switched off and enzymes were added into the hydrolysis reactor at t = 72 h. The expressions “in” and “out” represent the medium going from the filtration unit into the bioreactor and the
medium that leaves the bioreactor to the hydrolysis reactor, respectively.
purposes, such as ethanol production by fermentation. Therefore, it may be
advantageous to utilize other compounds than glucose for biomass formation.
By excluding the enzymatic decomposition, the glucose concentration was
sharply reduced, resulting in a higher uptake of xylose and acetic acid.
Combining the cultivation of Rhizopus sp. by SSFF with the addition of S.
cerevisiae in the hydrolysis reactor further reduced the glucose concentration
in the feed and increased the consumption of xylose, acetic acid, and ethanol.
The use of enzymatic hydrolysis and fermentation followed by distillation
prior to SSFF cultivation produced a result remarkably similar to the case
with the untreated slurry, i.e., without enzyme addition (Table 1).
In conclusion, reducing the glucose concentration in the present study
steered the uptake by Rhizopus sp. to xylose and acetic acid, which both can
be present as residual compounds in a biorefinery, without reducing either the
biomass yield or the specific growth rate.
3.4. Fermentation by Rhizopus sp.
Rhizopus sp. is also useful as a fermenting organism for ethanol
production and the combined production of valuable biomass and ethanol
is interesting in a biorefinery perspective. Performing a complete list of
process possibilities is beyond the scope of this study, and only the
production of ethanol and animal feed using Rhizopus sp. as producing
organism was investigated herein.
Two experiments were carried out, where Rhizopus sp. was initially
grown aerobically on straw hydrolysate in order to build up biomass. One
of the trials was stopped after 72 h (referred to in Table 1 as cultivation
without enzyme addition), and the biomass was harvested and measured
(4.4 g). The second experiment was initiated in a similar way, but after 72
h, cellulase (10 FPU/g SS) was added and the air supply to the
fermentation reactor was switched off (Fig. 4).
Please cite this article as: FazeliNejad S., Ferreira J.A., Brandberg T., Lennartsson P.R., Taherzadeh M.J. Fungal protein and ethanol from lignocelluloses
using Rhizopus pellets under simultaneous saccharification, filtration and fermentation (SSFF). Biofuel Research Journal 9 (2016) 372-378.
DOI: 10.18331/BRJ2016.3.1.7
.
378
FazeliNejad et al. / Biofuel Research Journal 9 (2016) 372-378
During the subsequent 94 h, the amount of biomass increased to 6.4 g,
suggesting a specific growth rate (µ) of 0.034/h during the anaerobic phase
(Table 1).
In the time span from 72 to 166 h, at least 11.5 g of ethanol was produced
(some may have evaporated), which would suggest an ethanol yield of 0.40
g/g consumed glucose and xylose, and an ethanol productivity of 0.023 g/g/h,
based on the average biomass concentration. The volumetric productivity of
ethanol, 0.24 g/L/h, was relatively low if compared with optimized
fermentations with S. cerevisiae (Balat, 2011). The xylose uptake
corresponded to only 5% of the consumed carbon source and the uptake
should be a sign of leakage of oxygen into the fermentation vessel,
considering that fungi normally do not consume xylose under anaerobic
conditions. According to the measurements, the concentration of xylose in the
hydrolysis vessel increased after 72 h, indicating the release of xylose from
the solid fraction as a result of the enzymatic decomposition.
3.5. Impact of inhibitors
After dilution to 5.0% SS, the concentrations of acetic acid, furfural, and
HMF were 1.8 g/L, 0.65 g/L, and 1.3 g/L, respectively. Considering a
previous study (FazeliNejad et al., 2013), these levels should not be very
inhibiting by themselves. It is worth quoting that hydrolysates of
lignocellulosic material usually contain other inhibitors as well, such as
phenolic compounds, whose concentrations were not measured in the present
study. It was observed in all the SSFF trials that ingoing furfural and HMF
were completely converted (i.e., not detected in the outflow) after 6-8 h of the
integrated fermentation, confirming in situ conversion of these compounds by
Rhizopus sp. (FazeliNejad et al., 2013). Furthermore, the specific growth rate,
µ, was similar in the hydrolysate-based SSFF experiments and the shake-flask
experiments with synthetic medium. It could thus be assumed that the impact
of inhibitors was limited.
4. Conclusions
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Rhizopus sp. in pellet form was successfully used for aerobic production of
biomass (animal feed) by SSFF from acid-pretreated wheat straw slurry with
biomass yields of up to 0.34 g biomass/g consumed monomeric sugars and
acetic acid. A surplus of glucose in the feed resulted in ethanol production
and reduced the biomass yield, whereas limiting glucose concentrations
resulted in higher consumption of xylose and acetic acid. The specific growth
rate was in the range of 0.013/h and 0.015/h and did not appear to be
influenced by the composition of the carbon source. Under anaerobic
conditions, an ethanol yield of 0.40 g/g and an ethanol productivity of 0.023
g/g/h were obtained using the Rhizopus sp. pellets. Overall, the present
strategy benefits from the easier separation of the biomass from the medium
and the fungus ability to assimilate carbon residuals in comparison with when
yeast is used. More specifically, it allows in situ separation of insoluble solids
and hence, a two-stage cultivation system practiced for production of
biomass and ethanol from whole stillage is not needed to be applied if
biomass is desired as a separate value-added product.
[20]
[21]
Acknowledgement
[22]
[16]
[17]
[18]
[19]
This work was financially supported by the Swedish Energy Agency.
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Please cite this article as: FazeliNejad S., Ferreira J.A., Brandberg T., Lennartsson P.R., Taherzadeh M.J. Fungal protein and ethanol from lignocelluloses
using Rhizopus pellets under simultaneous saccharification, filtration and fermentation (SSFF). Biofuel Research Journal 9 (2016) 372-378.
DOI: 10.18331/BRJ2016.3.1.7
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